1. Field of the Invention
The present invention relates to recombinant C-terminal .alpha.-amidating enzymes of Xenopus laevis origin and precursors thereof, DNAs coding for these polypeptides, plasmids containing the DNA, host cells transformed with the plasmid, a process for the production of the enzyme or precursor thereof using the transformed cells, and a process for the production of a C-terminal .alpha.-amidated peptide or protein using the enzyme.
2. Related Art
It is generally known that, in eukaryotic cells, some kinds of peptides or proteins are, after translation from a messenger RNA (mRNA), modified by an intracelular enzyme to mature to a natural-type peptide or protein (post-translational modification). But, since prokaryotic hosts such as E. coli, which are widely used to produce peptides or proteins of eukaryote origin, cannot carry out a post-translational modification of an expressed peptide or protein, it is sometimes difficult to directly produce a eukaryotic peptide or protein by a recombinant DNA technique using prokaryotic host cells.
One of this post-translational modification characteristic of eukaryotic cells of peptides or proteins is a modification reaction wherein an .alpha.-position of a carboxy terminal (C-terminal) of a peptide or protein is amidated, i.e., --COOH is converted to --CONH.sub.2, and it is known that many physiologically active peptides or proteins have been subjected to such modification. For example, as C-terminal .alpha.-amidated peptides, TRH(pGlu-His-Pro-NH.sub.2) and Caerulein ##STR1##
have been isolated, and a partial structure of precursors of these peptide determined from an analysis of the cDNA thereof. A general biosynthesis mechanism of such amidated peptides is understood to be that in which RNA is translated to a precursor of an amidated peptide, which is then amidated at the .alpha.-position of the C-terminal thereof by a C-terminal .alpha.-amidating enzyme. Note, in the above-mentioned reaction, the precursor of the C-terminal .alpha.-amidated peptide as a substrate for a C-terminal .alpha.-amidating enzyme is a peptide or protein represented by a general formula R-X-Gly, wherein R represents an amino acid sequence of the N-terminal side of the peptide or protein, X represents an amino acid residue which is to be .alpha.-amidated at the C-terminal thereof, and Gly represents a glycine residue.
It is known that, in some cases, the above-mentioned modification of peptide or protein is essential to the physiological activity thereof. For example, a conversion of the proline amide residue at the C-terminal of natural-type human calcitonin to proline residue decreases the physiological activity thereof to 1/1,600 of the original activity.
Because of the importance of clarifying the mechanism of .alpha.-amide formation in tissues, and the promising usefulness of the enzyme for the production of C-terminal .alpha.-amidated peptides using, for example, recombinant DNA techniques, many attempts to purify the enzyme have been made but the enzyme has not so far been obtained in a pure state. In porcine pituitary, Bradburg, A. F. et al, Nature 298, 686-688, 1982, first characterized the .alpha.-amidating activity converting a synthetic substrate D-Tyr-Val-Gly to D-Tyr-Val-NH.sub.2 , and demonstrated that the C-terminal glycine in the substrate serves as a nitrogen-donor for .alpha.-amidation. Eipper et al, Proc. Natl. Acad. Sci. US, 80, 5144-5148, 1983, reported that the .alpha.-amidating enzyme derived from the pituitary gland requires copper cation and ascorbate for its activity. Husain, I. et al, FEBS Lett., 152 227-281, 1983; and Kizer, J. S. et al, Proc. Natl. Acad. Sci. US, 81, 3228-3232, 1984, also reported a C-terminal .alpha.-amidating enzyme, but did not report a purified enzyme. Recently, Murthy A. S. N. et al, J. Biol. Chem., 261, 1815-1822, 1986, partially purified a C-terminal .alpha.-amidating enzyme from the pituitary gland of cattle, and showed that several types of enzymes having different molecular weights and electric charges are present. Nevertheless, no type of enzyme has been homogeneously purified.
Recently, Mizuno et al. succeeded in isolating a C-terminal .alpha.-amidating enzyme in a homogeneous and pure form from a skin of Xenopus laevis; see Mizuno, K et al, Biochem. Biophys. Res. Commun. 137, 984-991, 1988, and Japanese Patent Application No. 61-131089.
Nevertheless, the amount of the C-terminal .alpha.-amidating enzyme isolated from a skin of Xenopus laevis is limited, and not sufficient for use in the industrial production of C-terminal .alpha.-amidated peptides or proteins.